Genetic Engineering Technology Based On PD - 1 / PDL1 Signal Conversion Chimeric Receptor

While malignant tumor is still the leading cause of human death worldwide, adoptive cell transfer therapy is one of the most promising treatments besides traditional surgery, radiotherapy or chemotherapy. Adoptive cell transfer therapy uses genetically programmed, patient-derived lymphocytes transfected with chimeric immune receptors to recognize predefined tumor antigens with high specificity, resulting in successful tumor elimination. There are two common approaches:the heterodimeric TCR and chimeric antigen receptor (CAR).Recently, an increasing number of CARs targeting diverse tumors have been developed, and some of them have been tested in clinical trials. In these clinical trials, patient-derived lymphocytes were transfected with CAR targeting a specific tumor antigen and then adoptively transferred back to the patients, have successfully retarded tumor growth and lengthened patient survival. Unexpectedly, antigen-specific T cells usually encounter some negative regualtion in patients, so the T cells will lose the part or whole antitumor activity rather quickly. However, if tumor-infiltration lymphocyte are re-sorted and cultured in vitro for several days, their cellular function would recover. Such phenomenon suggests that immune inhibitory environment in vivo could impair the antitumor effect of T cells. Further research indicates these immune inhibitory effects originate from the tumor microenvironment, which consists of regulatory cells and inhibitory molecules. Therefore, the tumor microenvironment can partially or entirely block T cell activation, proliferation and killing activity by different mechanisms. Since tumor cells cannot be cleared completely, antitumor therapy would not be effective.PD-1/PDL1plays a key role in the tumor microenvironment, as it has been identified specifically as a mediocre prognostic indicator for several tumor types. Because it would block TCR signal and costimulatory signal, and inhibit T cell activation, proliferation and killing activity. Furthermore, such ligation also aids in the suppression of regulatory T cells and converts Thl cells into regulatory T cells. Meanwhile, T cell co-stimulatory receptors, such as CD28and41BB, remain absent in the tumor microenvironment. Therefore, is it possible to not only block PD-1/PDL1negative signal but also induce CD28and41BB positive signal by chimeric switch receptors? Is it possible to construct soluble form chimeric switch receptors in order to increase the efficiacy? Is it possible for chimeric switch receptors to resist both nTreg suppression and iTreg induction?In this study, we constructed both membrane-bound form and soluble form chimeric switch receptors. Later on, these switch receptors and CAR RNA were co-transduced into healthy human T cells to examine whether two form switch receptors could down-regulate the inhibition of PD-1/PDL1and also up-regulate the effect of T cells. In addition, we investigated the resistance of the both membrane-bound and soluble forms of the switch receptor against nTreg suppression. Moreover, three Treg in vitro inducing systems were established to test the ability of both forms of switch receptor to block iTreg induction. Our research would provide theoretical and experimental basis for removing the negative regulation CAR+T cells encountered in vivo, and offer a new strategy for optimal CAR-based adoptive transfer therapy.Part Ⅰ The expression and function of membrane-bound PD-1/PDL1chimeric switch receptorsIn the tumor microenvironment, the presence of inhibitory molecules and the absence of stimulatory molecules would lead to the blockade of T cell activation, proliferation and killing activity. Recently, a novel CTLA4-CD28chimera has been developed. Murine T cells retroviral transferred with CTLA4-CD28chimera showed enhanced cytokine production in vitro. Thus, adoptive transfer of gene-modified tumor antigen-specific T cells to tumor-bearing mice potentiated therapeutic efficacy of the T cells. Furthermore, another PD1-CD28chimera also has been developed, and this PD1-CD28chimera would also enhance T cell functions by increasing levels of ERK phosphorylation, cytokine secretion, and proliferation.In this experiment, we constructed two membrane-bound form chimeric switch receptors:PD1-CD28and PD1-41BB. After that, we made RNAs from these constructs and co-electroporated them with αCD19CAR RNA into stimulated T cells. Sixteen hours later, the expression of the switch receptors and CAR were detected by FACS. All the switch receptors and CAR had high expression on T cells. Later on, RNA-electroporated T cells were co-cultured with target cell groups of K562, K562-CD19or K562-CD19-PDL1at the ratio of1:4. The surface translocation of CD107a was measured after4hours. The result of this co-culturing showed that both PD1-CD28and PD1-41BB could increase the frequency of CD107a by resisting the PD-1/PDL1inhibitory signa1.Furthermore, RNA-electroporated T cells were also co-cultured with K562, K562-CD19, K562-CD19-PDL1, K562-meso-PDL1, Nalm6and Nalm6-PDL1at the ratio of1:1for16hours. The secretion of IL-2and IFN-y in the supernatant were collected and analyzed by ELISA assay. ELISA results showed that both PD1-CD28and PD1-41BB could induce the high-level secretion of IL-2and IFN-y via PD-1/PDL1signal in an antigen-specific manner.The above findings indicated the membrane-bound PD-1chimeric switch receptors not only express highly on T cells, but also block PD-1/PDL1inhibitory signal, as well as induce CD28or41BB stimulatory signal.Part II The expression and function of soluble PD-1/PDL1chimeric switch receptorsBi-specific T cell engager (BiTE) is based on single-chain bi-specific antibody to link two types of monoclonal antibodies so that BiTE can link two different epitopes from one or two cells and play the associated function. Classified as BiTE, a single-chain bi-specific antibody construct consists of aCD19and αCD3. It binds both CD3on T cells and CD19on tumor cells, shortens the distance between T cell and tumor cell, and activates TCR signaling to enhance T cell proliferation and killing activity. Thus, if we construct a BiTE with single-chain bi-specific antibody with specificity for PDL1 and CD28, would it be possible to shorten the distance of T cell and tumor cell, and also convert the negative signal from tumor cells to the positive signal for T cell activation?In our experiment, we selected three PDL1monoclonal antibodies (10A5,13G4and1B12) and one CD28monoclonal antibody (1412) to construct the soluble form chimeric switch receptors (pGEM-10A5-1412, pGEM-13G4-1412and pGEM-1B12-1412). We produced RNAs from these constructs and co-electroporated them with αCD19CAR RNA into stimulated T cells. Time points of4and16hours after electroporation were taken, and expression of the switch receptors on T cells was examined by FACS. The soluble switch receptors were harvested from the16-hour cultural supernatant and were used to stain K562-CD19-PDL1and K562-CD19tumor cells for30minutes. Afterwards, the FACS was performed to analyses the binding capacity of switch receptors on PDL1-expressing tumors or T cells, and results showed that all the switch receptors were capable binding both T cells and PDL1+tumor cells. In addition, these RNA-electroporated T cells were co-cultured with target cell groups of K562, K562-CD19or K562-CD19-PDL1at the ratio of1:4, and the surface translocation of CD107a was measured after4hours. The results indicated that10A5-1412and13G4-1412could increase the frequency of CD107a via resisting the PD-1/PDL1inhibitory signal. Moreover, these RNA-electroporated T cells were also put to co-culture with targets of K562, K562-CD19, K562-CD19-PDL1, K562-meso-PDL1, Nalm6and Nalm6-PDL1at the ratio of1:1for16hours. ELISA was carried out to detect the secretion of IL-2in the collected supernatant, and result suggested that only13G4-1412could induce the high-level secretion of IL-2via PDL1signal in an antigen-specific manner. Similar result of high level of IL-2secretion was also observed when10A5-1412or13G4-1412RNA electroporated T cells were co-cultured with K562-CD19cells. Moreover, when the PDL1on T cells or tumor cells or both was blocked by antibodies, the secretion of IL-2was decreased significantly.These data demonstrated the soluble PD-1/PDL1chimeric switch receptors not only have the capacity of binding T cells and tumor cells, but also blocking PD-1/PDL1inhibitory signal, as well as inducing CD28stimulatory signal. Thus, the soluble form chimeric switch receptors are much more sensitive than the membrane-bound form, since they could recognize low-level PDL1. Part Ⅲ The effect of PD-1/PDL1chimeric switch receptors on TregIn the tumor microenvironment, regulatory T cells (Treg) play a key role in suppressing the antitumor effect of adoptively transferred T cells, which becomes an obstacle for the adoptive transfer therapy. In prostate cancer, PD-1aids the suppression of regulatory T cells and even converts Thl cells into regulatory T cells. Yet blockage of PD-1/PDL1would impair the proliferative inhibition of Treg, increase antitumor associated cytokines and enhance antitumor activity. From the above results, switch receptors could block the PD-1/PDL1signal, so could they also block nTreg suppression and iTreg induction?To answer the question, nTregs, namely the CD4+CD25+T cells from PBMC of healthy human, were isolated. RNAs from switch receptors constructs and anti-her2/neu CAR were co-electroporated into CFSE Iabeled-CD4+CD25-T cells, which would be stimulated with inactivated K562-her2/neu tumor cells, and co-cultured with different doses of CD4+CD25+T cells. After6or8days, the proliferation of CD4+CD25-T cells was detected by FACS. It was found that two forms of switch receptors could resist the proliferative inhibition of nTreg.To further examine whether switch receptors could block the process of iTreg induction, we first established three Treg in vitro inducing systems to induce Naive T cells into iTreg with immune suppression. Then, RNAs from switch receptor constructs were electroporated into Naive T cells, which were induced by three different inducing systems for5days. However, we found that only the soluble form switch receptor10A5-1412could partially block the process of Treg induction.In summary, we constructed membrane-bound PD-1/PDL1chimeric switch receptors and successfully constructed soluble PD-1/PDL1chimeric switch receptors for the first time. With the first signal from CAR, both membrane-bound form and soluble form of switch receptors block PD1/PDL1negative signal, induce CD28or41BB positive signal, and resist nTreg suppression. Moreover, soluble chimeric switch receptor13G4-1412could partially block iTreg induction. Thus, this study might provide a new strategy for optimal adoptive CAR+T cells transfer therapy and show a promising perspective in clinical application.